Device and method for the detection of an analyte utilizing mesoscale flow systems

ABSTRACT

Disclosed are devices for detecting the presence of a preselected analyte in a fluid sample. The devices comprise a substrate microfabricated to define a sample inlet port, and a mesoscale flow system that includes a sample flow channel extending from the inlet port. The mesoscale flow system further includes an analyte detection region in fluid communication with the flow channel comprised of a binding moiety for specifically binding the analyte. The detection region is constructed with a mesoscale dimension sufficiently small to enhance binding of the binding moiety and the analyte. The binding moiety may be immobilized in the detection region. The mesoscale detection systems of the invention may be used in a wide range of applications, including the detection of cells or macromolecules, or for monitoring reactions or cell culture growth.

REFERENCE TO RELATED APPLICATIONS

This is a continuation of U.S. patent application Ser. No. 10/348,438,filed Jan. 21, 2003, which is a continuation of U.S. patent applicationSer. No. 09/237,523, filed Jan. 27, 1999, now U.S. Pat. No. 6,551,841,which is a continuation of U.S. patent application Ser. No. 08/811,873,filed Mar. 5, 1997, now U.S. Pat. No. 5,866,345, which is a continuationof U.S. patent application Ser. No. 08/347,498, filed Nov. 30, 1994, nowU.S. Pat. No. 5,637,469, which is a continuation of U.S. patentapplication Ser. No. 07/877,702, filed May 1, 1992, now abandoned. Theentire disclosure of each of the aforementioned applications isincorporated by reference in the present application.

BACKGROUND OF THE INVENTION

This invention relates generally to methods and apparatus for conductinganalyses. More particularly, the invention relates to the design andconstruction of small, typically single-use, modules capable ofreceiving and rapidly conducting a predetermined assay protocol on afluid sample.

In recent decades the art has developed a very large number ofprotocols, test kits, and cartridges for conducting analyses onbiological samples for various diagnostic and monitoring purposes.Immunoassays, agglutination assays, and analyses based on polymerasechain reaction, various ligand-receptor interactions, and differentialmigration of species in a complex sample all have been used to determinethe presence or concentration of various biological compounds orcontaminants, or the presence of particular cell types.

Recently, small, disposable devices have been developed for handlingbiological samples and for conducting certain clinical tests. Shoji etal. reported the use of a miniature blood gas analyzer fabricated on asilicon wafer. Shoji et al., Sensors and Actuators, 15:101–107 (1988).Sato et al. reported a cell fusion technique using micromechanicalsilicon devices. Sato et al., Sensors and Actuators, A21–A23:948–953(1990). Ciba Corning Diagnostics Corp. (USA) has manufactured amicroprocessor-controlled laser photometer for detecting blood clotting.

Micromachining technology originated in the microelectronics industry.Angell et al., Scientific American, 248:44–55 (1983). Micromachiningtechnology has enabled the manufacture of microengineered devices havingstructural elements with minimal dimensions ranging from tens of microns(the dimensions of biological cells) to nanometers (the dimensions ofsome biological macromolecules). This scale is referred to herein as“mesoscale”. Most experiments involving mesoscale structures haveinvolved studies of micromechanics, i.e., mechanical motion and flowproperties. The potential capability of mesoscale structures has notbeen exploited fully in the life sciences.

Brunette (Exper. Cell Res., 167:203–217 (1986) and 164:11–26 (1986))studied the behavior of fibroblasts and epithelial cells in grooves insilicon, titanium-coated polymers and the like. McCartney et al. (CancerRes., 41:3046–3051 (1981)) examined the behavior of tumor cells ingrooved plastic substrates. LaCelle (Blood Cells, 12:179–189 (1986))studied leukocyte and erythrocyte flow in microcapillaries to gaininsight into microcirculation. Hung and Weissman reported a study offluid dynamics in micromachined channels, but did not produce dataassociated with an analytic device. Hung et al., Med. and Biol.Engineering, 9:237–245 (1971); and Weissman et al., Am. Inst. Chem. Eng.J., 17:25–30 (1971). Columbus et al. utilized a sandwich composed of twoorthogonally orientated v-grooved embossed sheets in the control ofcapillary flow of biological fluids to discrete ion-selective electrodesin an experimental multi-channel test device. Columbus et al., Clin.Chem., 33:1531–1537 (1987). Masuda et al. and Washizu et al. havereported the use of a fluid flow chamber for the manipulation of cells(e.g. cell fusion). Masuda et al., Proceedings IEEE/IAS Meeting, pp.1549–1553 (1987); and Washizu et al., Proceedings IEEE/IAS Meeting pp.1735–1740 (1988). The art has not fully explored the potential of usingmesoscale devices for the analyses of biological fluids and detection ofmicroorganisms.

The current analytical techniques utilized for the detection ofmicroorganisms are rarely automated, usually require incubation in asuitable medium to increase the number of organisms, and invariablyemploy visual and/or chemical methods to identify the strain orsub-species. The inherent delay in such methods frequently necessitatesmedical intervention prior to definitive identification of the nature ofan infection. In industrial, public health or clinical environments,such delays may have serious consequences. There is a need forconvenient systems for the rapid detection of microorganisms.

An object of the invention is to provide analytical systems with optimalreaction environments that can analyze microvolumes of sample, detectsubstances present in very low concentrations, and produce analyticalresults rapidly. Another object is to provide easily mass produced,disposable, small (e.g., less than 1 cc in volume) devices havingmesoscale functional elements capable of rapid, automated analyses ofpreselected molecular or cellular analytes, in a range of biological andother applications. It is a further object of the invention to provide afamily of such devices that individually can be used to implement arange of rapid clinical tests, e.g., tests for bacterial contamination,virus infection, sperm motility, blood parameters, contaminants in food,water, or body fluids, and the like.

SUMMARY OF THE INVENTION

The invention provides methods and devices for the detection of apreselected analyte in a fluid sample. The device comprises a solidsubstrate, typically on the order of a few millimeters thick andapproximately 0.2 to 2.0 centimeters square, microfabricated to define asample inlet port and a mesoscale flow system. The term “mesoscale” isused herein to define chambers and flow passages having cross-sectionaldimensions on the order of 0.1 μm to 500 μm. The mesoscale flow channelsand fluid handling regions have a preferred depth on the order of 0.1 μmto 100 μm, typically 2–50 μm. The channels have preferred widths on theorder of 2.0 μm to 500 μm, more preferably 3–100 μm. For manyapplications, channels of 5–50 μm widths will be useful. Chambers in thesubstrates often will have larger dimensions, e.g., a few millimeters.

The mesoscale flow system of the device includes a sample flow channel,extending from the inlet port, and an analyte detection region in fluidcommunication with the flow channel. The analyte detection region isprovided with a binding moiety, optionally immobilized therewithin, forspecifically binding the analyte. The mesoscale dimension of thedetection region kinetically enhances binding of the binding moiety andthe analyte. That is, in the detection region, reactants are broughtclose together in a confined space so that multiple molecular collisionsoccur. The devices may be used to implement a variety of automated,sensitive and rapid clinical tests including the analysis of cells ormacromolecules, or for monitoring reactions or cell growth.

Generally, as disclosed herein, the solid substrate comprises a chipcontaining the mesoscale flow system. The chips are designed to exploita combination of functional geometrical features and generally knowntypes of clinical chemistry to implement the detection ofmicroquantities of an analyte. The mesoscale flow system may be designedand fabricated from silicon and other solid substrates using establishedmicromachining methods, or by molding polymeric materials. The mesoscaleflow systems in the devices may be constructed by microfabricating flowchannel(s) and detection region(s) into the surface of the substrate,and then adhering a cover, e.g., a transparent glass cover, over thesurface. The channels and chambers in cross-section taken through thethickness of the chip may be triangular, truncated conical, square,rectangular, circular, or any other shape. The devices typically aredesignated on a scale suitable to analyze microvolumes (<5 μL) ofsample, introduced into the flow system through an inlet port defined,e.g., by a hole communicating with the flow system through the substrateor through a transparent coverslip. Cells or other analytes present invery low concentrations (e.g. nanogram quantities) in microvolumes of asample fluid can be rapidly analyzed (e.g., <10 minutes).

The chips typically will be used with an appliance which contains anesting site for holding the chip, and which mates an input port on thechip with a flow line in the appliance. After biological fluid such asblood, plasma, serum, urine, sputum, saliva, or other fluids suspectedto contain a particular analyte, cellular contaminant, or toxin isapplied to the inlet port of the substrate, the chip is placed in theappliance and a pump is actuated to force the sample through the flowsystem. Alternatively, a sample may be injected into the chip by theappliance, or the sample may enter the mesoscale flow system of the chipthrough the inlet port by capillary action.

In the devices, the binding of an analyte to a binding moiety serves asa positive indication of the presence of the analyte in a sample. Themesoscale detection region is provided with a binding moiety capable ofspecifically binding to the preselected analyte. The binding moiety maybe delivered to the detection region in, e.g., a solution.Alternatively, the binding moiety may be immobilized in the detectionregion. The internal surfaces of the mesoscale detection region of thedevice may be coated with an immobilized binding moiety to enable thesurface to interact with a fluid sample in order to detect or separatespecific fluid sample constituents. Antibodies or polynucleotide probesmay be immobilized on the surface of the flow channels, enabling the useof the mesoscale flow systems for immunoassays or polynucleotidehybridization assays. The binding moiety also may comprise a ligand orreceptor. A binding moiety capable of binding cells via a cell surfacemolecule may be utilized, to enable the isolation or detection of a cellpopulation in a biological microsample. The mesoscale flow system mayalso include protrusions or a section of reduced cross sectional area toenable the sorting or lysis of cells in the microsample upon flowthrough the flow system.

Analyte binding to a binding moiety in the detection region may detectedoptically, e.g., through a transparent or translucent window, such as atransparent cover over the detection region or through a translucentsection of the substrate itself. Changes in color, fluorescence,luminescence, etc., upon binding of the analyte and the binding moiety,indicating a positive assay can be detected either visually or bymachine. The appliance may include sensing equipment, such as aspectrophotometer, capable of detecting changes in optical properties,due to the binding of an analyte to a binding moiety in the detectionregion, through a clear cover disposed over the detection region.

The device may further include means for delivering reagents such as alabeled substance to the detection region that binds to the analyte toprovide a detectable signal indicative of the presence of the analyte.Optionally, depending on the protocol being exploited in the structureof the chip, the appliance also may be designed to inject reagentsnecessary to complete the assay, e.g., to inject a binding proteintagged with an optically detectable moiety, a substrate solution forreaction with an enzyme, or other reagents.

A positive assay may also be indicated by detectable agglutination orflow impedance upon analyte binding. The presence of a preselectedanalyte in a fluid sample may be detected by sensing analyte-inducedchanges in sample fluid flow properties, such as changes in the pressureor electrical conductivity, at different points in the flow system. Inone embodiment, analyte induced restriction or blockage of flow in themesoscale flow system, e.g., in the fractal region, may be detected bypressure detectors, e.g., in the appliance used in combination with thedevice. In another embodiment, analyte-induced changes in conductivityin a region of the flow system caused by introduction of a sample fluidmay be readily detected through electrical conductivity sensors incontact with the flow system. For example, the presence of analyte maycause clogging of a restricted flow passage, and beyond the passage, theabsence of liquid can be detected by measuring conductivity. Theappliance also may include electrical contacts in the nesting regionwhich mate with contacts integrated into the structure of the chip to,e.g., provide electrical resistance heating or cooling to a portion ofthe flow system, or receive electrical signals indicative of a pressurereading, conductivity, or the like, sensed in some region of the flowsystem to indicate (flow restriction, as a) positive indication of thepresence of the analyte.

The mesoscale devices can be adapted to perform a wide range ofbiological or other tests. A device may include two or more separatedflow systems, e.g., fed by a common inlet port, with different bindingmoieties in, e.g., different detection regions to enable the detectionof two or more analytes simultaneously. The device may also comprise acontrol flow system so that data from the sample region and the controlregion may be detected and compared. Essentially any test involvingdetection of the presence or concentration of a molecular or atomicscale analyte, or the presence of a particular cell type, can beimplemented to advantage in such structures. The mesoscale devices mayprovide a rapid chemical test for the detection of pathogenic bacteriaor viruses. The devices may also provide a rapid test for the presenceor concentration of blood constituents such as hormones. Otherapplications include but are not limited to a range of other biologicalassays such as blood type testing.

The devices as disclosed herein are all characterized by a mesoscaledetection region containing a binding moiety that reacts with theanalyte component, such as a molecular analyte or a cell type, to detectthe presence or concentration of the analyte. The device may be readilysterilized prior to an assay. Assays may be completed rapidly, and atthe conclusion of the assay the chip can be discarded, whichadvantageously prevents contamination between samples, entombspotentially hazardous material, produces only microvolumes of wastefluid for disposal, and provides an inexpensive, microsample analysis.Some of the features and benefits of the devices are summarized in Table1.

TABLE 1 Feature Benefit Flexibility No limits to the number of chipdesigns or applications available. Reproducible Allows reliable,standardized, mass production of chips. Low Cost Allows competitivepricing with Production existing systems. Disposable nature forsingle-use processes. Small Size No bulky instrumentation required.Lends itself to portable units and systems designed for use in non-conventional lab environments. Minimal storage and shipping costs.Microscale Minimal sample and reagent volumes required. Reduces reagentcosts, especially for more expensive, specialized test procedures.Allows simplified instrumentation schemes. Sterility Chips can besterilized for use in microbiological assays and other proceduresrequiring clean environments. Sealed System Minimizes biohazards.Ensures process integrity. Multiple Circuit Can perform multipleprocesses or Capabilities analyses on a single chip. Allows panelassays. Multiple Expands capabilities for assay and Detector processmonitoring to virtually any Capabilities system. Allows broad range ofapplications. Reusable Chips Reduces per process cost to the user forcertain applications.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic longitudinal cross sectional view of a deviceaccording to the invention that includes a solid substrate 14, on whichare machined entry ports 16 connected by mesoscale flow channel 20, witha transparent cover 12 adhered to the surface of the substrate.

FIG. 2 is a perspective view of the device of FIG. 1.

FIG. 3 is a cross sectional view of a support block 30 for holdingdevice 10 that includes ports 32 for delivery or removal of reagents orsample fluids from device 10.

FIG. 4 is a schematic plan view of a substrate 14 fabricated with afractally bifurcating system of flow channels 40 symmetrically disposedon the substrate.

FIG. 5 is a schematic illustration of analytical device 10 nested withinappliance 50, which is used to support the device 10 and to regulate anddetect the pressure of sample fluids in device 10.

FIG. 6 is a schematic illustration of a top view of a device comprisinga substrate 14, microfabricated with a pair of mesoscale flow systemswhich include inlet ports 16 and flow channel 20.

FIG. 7 is a schematic illustration of a top view of another devicecomprising substrate 14, fabricated with a mesoscale flow system thatincludes inlet ports 16, flow channel 20 and sample detection chamber22.

FIG. 8 is a schematic illustration of a top view of a solid substrate 14microfabricated with three flow paths 20 each of which defines a pair ofdetection chambers 22 and 24. Chambers 22A, 22B and 22C containantibodies to group A blood antigen, group B blood antigen and Rhesusantigen respectively, while chambers 24A, 24B and 24C are controlchambers.

FIG. 9 is a schematic illustration of a top view of a solid substrate 14microfabricated with three sample detection chambers 22A, 22B and 22Ccontaining beads on which are immobilized antibodies to group A bloodantigen, group B blood antigen and Rhesus antigen respectively.

FIGS. 10A–D are schematic illustrations of a cross-section of a portionof a mesoscale flow channel 20 within a substrate 14, on whichantibodies 103 are immobilized, and illustrating changing states of thesystem during an analysis.

FIGS. 11A–D are schematic illustrations of a cross-section of a portionof a mesoscale flow channel 20 within a substrate 14, on which DNAbinding probes 110 are immobilized, and illustrating changing states ofthe system during an analysis.

FIG. 12 is a cross sectional perspective view of a flow channel 20 onthe inert substrate 14 with cell or debris filtering protrusions 122extending from a wall of the flow channel.

FIG. 13 is a cross sectional view of a flow channel 20 on the inertsubstrate 14 with cell piercing protrusions 124 extending from a wall ofthe channel.

FIG. 14 is a schematic plan view of a sperm function testing apparatusconstructed in accordance with the invention.

FIG. 15 is a schematic plan view of a mesoscale PCR analytical deviceconstructed in accordance with the invention.

FIG. 16 is a schematic top view of a analytical device fabricated with aseries of mesoscale chambers suitable for implementing a variety offunctions including cell sorting, cell lysing and PCR analysis.

FIG. 17 a is a schematic longitudinal cross sectional view of a deviceaccording to the invention which includes electrical contacts 17 and 18for measuring conductivity of fluids in the device.

FIG. 17 b is a perspective view of the device shown in FIG. 17 a.

FIG. 18 is a schematic plan view of a substrate microfabricated with apair of fractally bifurcating flow channels 40.

FIG. 19 is a schematic perspective view of an apparatus 60 used incombination with device 10 for viewing the contents of device 10.

FIG. 20 is a schematic cross sectional view of the apparatus 60 of FIG.19.

FIG. 21 is a schematic plan view of device 10 microfabricated with amesoscale flow system that includes the tortuous channels 22A and 22Bwhich allow the timed addition and mixing of assay components during anassay.

Like reference characters in the respective drawn figures indicatecorresponding parts.

DETAILED DESCRIPTION

The invention provides a family of small, mass produced, typicallyone-use devices for detecting a particular analyte in a fluidmicrosample. The device comprises a solid substrate, typically on theorder of a few millimeters thick and approximately 0.2 to 2.0centimeters square, that is microfabricated to define a sample inletport and a mesoscale flow system. The mesoscale flow system includes atleast one sample flow channel extending from the inlet port and at leastone analyte detection region in fluid communication with the flowchannel which contains a binding moiety for specifically binding theanalyte. optionally the binding moiety may be immobilized within thedetection region. As disclosed herein, mesoscale detection systems maybe used in a wide range of rapid tests, including the analysis of cellsor macromolecules, or for monitoring reactions or cell culture growth.The devices may be fabricated with two or more mesoscale flow systemswhich comprise two or more different detection regions containingbinding moieties for different analytes, allowing two or more assays tobe conducted simultaneously. At the conclusion of the assay the devicestypically are discarded.

Mesoscale devices having flow channels and chambers with at least onemesoscale dimension can be designed and fabricated in large quantitiesfrom a solid substrate material. Silicon is preferred because of theenormous body of technology permitting its precise and efficientfabrication, but other materials may be used including-polymers such aspolytetrafluoroethylenes. The sample inlet port, the mesoscale flowsystem, including the sample flow channel(s) and the analyte detectionregion(s), and other functional elements thus may be fabricatedinexpensively in large quantities from a silicon substrate by any of avariety of micromachining methods known to those skilled in the art. Themicromachining methods available include film deposition processes suchas spin coating and chemical vapor deposition, laser fabrication orphotolithographic techniques such as UV or X-ray processes, or etchingmethods including wet chemical processes or plasma processes. (See,e.g., Manz et al., Trends in Analytical Chemistry 10: 144–149 (1991)).Flow channels of varying widths and depths can be fabricated withmesoscale dimensions, i.e., with cross-sectional dimensions on the orderof 0.1 to 500 μm.

The silicon substrate containing a fabricated mesoscale flow channel maybe covered and sealed with a thin anodically bonded glass cover. Otherclear or opaque cover materials may be used. Alternatively, two siliconsubstrates can be sandwiched, or a silicon substrate may be sandwichedbetween two glass covers. The use of a transparent cover results in awindow which facilitates dynamic viewing of the channel contents andallows optical probing of the detection region either visually or bymachine. Other fabrication approaches may be used. In one embodiment,electron micrographs of biological structures such as circulatorynetworks may be used as masks for fabricating mesoscale flow systems onthe substrate. Mesoscale flow systems may be fabricated in a range ofsizes and conformations.

In one embodiment, illustrated schematically in FIGS. 1 and 2, thedevice 10 may include a silicon substrate 14 microfabricated with amesoscale flow channel 20, which, in this instance, also serves as adetection region, and which may be provided with binding moietiescapable of binding a preselected analyte. Sample or reagent fluid may beadded or recovered from flow channel 20 via ports 16 which arefabricated on either end of the flow channel 20. The substrate 14 iscovered with a glass or plastic window 12. During an analysis, thedevice 10 may be placed in support structure 30 (FIG. 3), which isprovided with internal flow paths 32 for delivery and recovery of samplefluid through the inlet ports of device 10. The dimensions of themicrochannels in the silicon mesoscale devices may vary in the rangefrom approximately 2.0 μm–500 μm wide and approximately 0.1 μm–500 μm indepth, a range comparable to cellular or macromolecular dimensions,where fluid motion of multiphasic materials such as fluid and cellculture medium has not been systemically investigated. The inlet portson the devices may be microfabricated with mesoscale or, alternatively,larger dimensions.

The capacity of the devices is very small and therefore reduces theamount of sample fluid required for an analysis. For example, in a 1cm×1 cm silicon substrate, having on its surface an array of 500 grooveswhich are 10 microns wide×10 microns deep×1 cm (10⁴ microns) long, thevolume of each groove is 10⁻³ μL and the total volume of the 500 groovesis 0.5 μL. The low volume of the mesoscale flow systems enhances thereaction rates of assays conducted in the devices. For example, in amesoscale detection chamber containing a surface coating of animmobilized binding moiety, as predicted by the Law of Mass Action, asthe volume of the mesoscale detection chamber decreases, the surfacearea to volume ratio of the binding moiety in the detection regionincreases, which results in an increased rate of intermolecular reactionbetween the analyte and the binding moiety. The entire mesoscale flowsystems of devices of the invention typically have volumes on the orderof less than 10 μL. Detection chambers are small enough in at least onedimension to favor fast kinetics. The mesoscale flow systems in thedevices may be microfabricated with microliter volumes, or alternativelynanoliter volumes or less, which advantageously limits the amount ofsample and/or reagent fluids required for an assay.

The analytical devices containing the mesoscale flow system can be usedin combination with an appliance for delivering and receiving fluids toand from the devices, such as appliance 50, shown schematically in FIG.5, which incorporates a nesting site 58 for holding the device 10, andfor registering ports, e.g., ports 16 on the device 10, with a flow line56 in the appliance. The appliance may include means, such as pump 52shown in FIG. 5, for forcing the sample through the flow system. After abiological fluid sample suspected to contain a particular analyte isapplied to the inlet port 51 of the appliance, pump 52 is actuated toforce the sample into port 16 of device 10 and the mesoscale flowchannel 20. Alternatively a sample may be injected into the chip by theappliance, or the sample may enter the mesoscale flow system of thedevice through the inlet port by capillary action. In anotherembodiment, the appliance may be disposed over the substrate, and may beprovided with a flow line communicating with the inlet ports in thedevice, e.g., in the absence of a cover over the device, to enable asample to be injected via the appliance into the device. Otherembodiments of appliances may be fabricated in accordance with theinvention for use in different assay protocols with different devices.The flow systems of the devices may be filled to a hydraulically fullvolume and the appliance may be utilized to direct the flow of fluidthrough the flow system, e.g., by means of valves located in the deviceor in the appliance.

The analytical devices also may be utilized in combination with anappliance for viewing the contents of the mesoscale channels in thedevices. The appliance in one embodiment may comprise a microscope forviewing the contents of the mesoscale channels in the devices. Inanother embodiment, a camera may be included in the appliance, asillustrated in the appliance 60 shown schematically in FIGS. 19 and 20.The appliance 60 is provided with a housing 62, a viewing screen 64 anda slot 66 for inserting a chip into the appliance. As shown in crosssection in FIG. 20, the appliance 60 also includes a video camera 68, anoptical system 70, and a tilt mechanism 72 for holding device 10, andallowing the placement and angle of device 10 to be adjusted manually.The optical system 70 may include a lens system for magnifying thechannel contents, as well as a light source. The video camera 68 andscreen 64 allow analyte induced changes in sample fluid properties, suchas flow properties or color, to be monitored visually, and optionallyrecorded using the appliance.

Binding moieties may be introduced into the mesoscale detection regionin a solution via an inlet port in fluid communication with thedetection region. Alternatively, binding moieties may be immobilized inthe mesoscale detection region of the analytical devices after itsmanufacture by, for example, physical absorption or chemical attachmentto the surface of the flow system or to a solid phase reactant such as apolymeric bead disposed in the detection region.

The surfaces of the mesoscale detection channels in the siliconsubstrates can be chemically activated and reacted with a protein,lipid, polysaccharide or other macromolecule to form a coated surface inthe mesoscale flow channels. Techniques for the chemical activation ofsilaceous surfaces are available in the art. (See, e.g., Haller in:Solid Phase Biochemistry, W. H. Scouten, Ed., John Wiley, New York, pp535–597 (1983); and Mandenius et al., Anal. Biochem., 137:106–114 (1984)and 170: 68–72 (1988) and Mandenius et al., Methods in Enzymology, 137:388–394). There are a number of techniques in the art for attachingbiomolecules to silicon. For example, enzymes may be immobilized onsilicon devices via entrapment in a photo-crosslinkable polyvinylalcohol (Howe et al., IEEE Transactions Electron Devices, ED33:499–506(1986) or attached indirectly using preformed membranes (Hanazato etal., IEEE Transactions Electron Devices, ED33:47–51 (1986). Ahydrophobic bilayer glycerol monooleate coating may be fabricated on asilicon substrate. Fromherz et al., Biochim. Biophys. Acta, 1062:103–107(1991).

Protein conjugation and immobilization techniques known in the art maybe adapted for use with activated silaceous surfaces. Kennedy et al.,Clin. Chem. Acta, 70:1–31 (1976); Sankolli et al., J. Imm. Methods,104:191–194 (1987); Kricka et al., Clin. Chem., 26:741–744 (1980); andDeLuca et al., Arch. Biochem. Biophys., 225:285–291 (1983). Knownchemistries in the art may be adapted for use in attaching biomoleculesto coated or uncoated silicon channel surfaces. A binding moiety such asan antigen binding protein, a polynucleotide probe, or one of aligand/receptor pair may be attached to the silicon channel surfaces.The surface coated mesoscale flow systems can be utilized in any of awide range of available binding assays known in the art such asimmunoassays, enzymatic assays, ligand/binder assays, polynucleotidehybridization assays, and cell surface binding assays. The detection ofcellular or macromolecular analytes can be implemented by selecting theappropriate binding moiety coated on the surface of the detectionregion.

In addition, magnetic beads may be utilized in the device, which can bemoved through the mesoscale flow system using an externally appliedmagnetic field, e.g., from a magnetic source located in an applianceutilized in combination with the device. The binding moiety or otherreagent required in an assay may be immobilized on a magnetic bead toenable, e.g., the delivery of the binding moiety to the detection regionto bind to the analyte. After the binding of the analyte to the bindingmoiety attached to the magnetic bead, the analyte may be, e.g., furtherpurified, or moved via an external magnetic field to a differentdetection region in the flow system for further analyses.

The binding of the analyte to the binding moiety in the detection regioncan be detected by any of a number of methods including monitoring thepressure or electrical conductivity of sample fluids in the device asdisclosed herein or by optical detection through a transparent covereither visually or by machine. Devices such as valves, mesoscalepressure sensors, and other mechanical sensors can be directlyfabricated on the silicon substrate and can be mass-produced accordingto well established technologies. Angell et al., Scientific American248:44–55 (1983).

The binding of an analyte to a binding moiety in the detection regioncan be detected optically. The simplest embodiment is one in which apositive result is indicated by an agglomeration or agglutination ofparticles, or development of or change in color, which can be visuallyobserved, optimally with the aid of a microscope. The optical detectionof the binding of an analyte to a binding moiety in the mesoscaledetection chambers can be implemented by the attachment of a detectablelabel, such as a fluorescent or luminescent molecule or polymericsupport, such as a bead, to either the analyte or the binding moietyusing assay protocols known per se. The luminescent or fluorescent labelin the detection region can be detected by light microscopy through atranslucent window disposed over the substrate. Analytes may be detectedby a luminescent or fluorescent signal produced by a binding moiety uponbinding of the analyte. Alternatively, a second labelled substance, suchas a fluorescent labelled antibody can be delivered through the flowsystem to bind to the bound analyte/binding moiety complex in thedetection region to produce a “sandwich” including an opticallydetectable moiety whose presence is indicative of the presence of theanalyte. For example, immunogold or immunofluorescent labels reported inthe prior art may be utilized. (See, e.g., Rosenberg et al., Clin. Chem.30: 1462–1466 (1984); Rosenberg et al., Clin. Chem. 31: 1444–1448(1985); and Goin et al., Clin. Chem. 32: 1655–1659 (1986)).

The binding of an analyte in a liquid biological fluid sample to abinding moiety in the detection region also may be detected by sensingelectrical conductivity at some region within the device. Theconductivity of liquid in the mesoscale flow paths can be measured inorder to detect changes in electrical properties upon analyte binding tobinding moieties in the detection region. The conductivity may bemeasured, e.g., in the device 10 shown schematically in FIGS. 17 a and17 b. Device 10 includes the silicon substrate 14 on which aremicrofabricated inlet ports 16 and flow channel 20. The substrate iscovered by a translucent window 12. Electrical conductivity measurementsare made using the electrical contacts 18 which are fabricated on thetop side of the substrate in contact with the mesoscale sample flowchannel 20, and which are connected to contacts 17 which extend throughto the bottom of the substrate. The contacts 17 can be fabricated byknown techniques of thermal gradient zone melting. (See Zemel et al.,in: Fundamentals and Applications of Chemical Sensors, D. Schuetzle andR. Hammerle, Eds., ACS Symposium Series 309, Washington, D.C., 1986, p.2.) Device 10 may be nested in an appliance such as appliance 50, shownin FIG. 5, capable of detecting conductivity changes through thecontacts 17. Changes in conductivity can be correlated with changes influid properties, such as fluid pressure, induced by analyte binding inthe detection region.

The binding of an analyte to a binding moiety in the detection regionalso can be detected by monitoring the pressure of the sample fluids incertain specially designed regions of the mesoscale flow passages. Forexample, a pressure detector connected to sample fluid entering andexiting the mesoscale flow system will allow the detection of pressuredecreases caused by analyte binding and resulting clogging or flowrestriction. FIG. 5 shows schematically, as an examples device 10, whichis nested within appliance 50, which includes two pressure detectors 54for detecting flow pressure of fluids entering and exiting device 10through ports 16. When, during an assay, particles agglomerate ormolecules chemically interact to form a network clogging the flowpassage or increasing the viscosity of the liquid, that change can bedetected as a pressure change indicative as a positive result. Amesoscale pressure sensor also may be fabricated directly on the siliconsubstrate. Angell et al., Scientific American 248: 44–55 (1983).

This form of detection of an analyte binding to a binding moiety in thedetection region can be enhanced by geometries sensitive to flowrestriction in the flow system. In one embodiment, the mesoscale flowchannels in the devices may be constructed with a “fractal” pattern,i.e., of a pattern of serially bifurcating flow channels. FIG. 18illustrates schematically one embodiment of a device 10 which includessubstrate 14 microfabricated with two fractal flow systems 40. Thefractally bifurcating channels may be fabricated on a silicon substratewith reduced dimensions at each bifurcation, providing sequentiallynarrower flow channels, as illustrated schematically in FIG. 4. FIG. 4shows a schematic plan view of a substrate 14 fabricated with afractally bifurcating system of flow channels 40 connected to ports 16.The channels in this embodiment are symmetrically disposed and have asequentially narrower diameter towards the center of the substrate.Fluid flow in these fractally constructed flow systems is very sensitiveto fluid viscosity and to the development of flow restriction caused,for example, by the proliferation of cells, or the agglomeration ofcells, particles, or macromolecular complexes that may be present in asample. The detection of the presence of an analyte based on flowrestriction is described in U.S. Ser. No. 07/877,701, the disclosure ofwhich is incorporated herein by reference.

The fractally designed microchannels readily allow, e.g., the growth oforganisms in a culture to be monitored on the basis of flow impedancedue to changes in fluid viscosity which can be detected, e.g., opticallythrough a transparent cover over the substrate. The presence and growthof an organism in a sample will influence the flow characteristicswithin the fractal. One or more pressure sensors may be utilized todetect pressure changes due to changes in fluid properties caused by thepresence of an analyte in or beyond the fractal flow paths. Changes inconductivity upon analyte binding also may be readily detected throughelectrical conductivity sensors in contact with the flow region. Forexample, clogging of the fractal region 40 of device 10 in FIG. 4, whichblocks flow of analyte from input port 16A to outlet port 16B may bedetected by a conventional conductivity probe 17, whose output isindicative of the presence or absence of aqueous fluid in the outflowchannel. Binding moieties may be provided in fractal region, e.g.,immobilized on the surface of the fractal flow path, or on a solid phasereactant such as a bead, to bind to the analyte and enhance flowrestriction in the fractal flow path.

A large number of binding assay protocols known in the art may beexploited in the mesoscale detection systems of the invention.

The reaction of an analyte with a binding moiety in the detection regionmay be detected by means of an agglutination. A fluorescent orluminescent labelled molecule or bead capable of binding to the analyteor analyte/binding moiety complex in the detection region may be used toenable the detection of agglutination of the binding moiety and theanalyte by light microscopy through a translucent cover over thedetection region. For example, the agglutination of blood cells in amesoscale detection chamber can serve as a positive test for the bloodtype of the sample. Antibodies may be coated, either chemically or byabsorption, on the surface of the detection region to induceagglutination, giving a positive test for blood type. The blood samplemay be mixed with a fluorescent dye to label the blood cells and toenable the optical detection of the agglutination reaction. Antibodiesbound to fluorescent beads also may be utilized. A plurality ofdetection regions housing different antibodies may be fabricated in themesoscale flow paths to allow the simultaneous assay of e.g., A, B andRh blood types in one device.

Immunochemical assay techniques known in the art, such as antibodysandwich assays and enzyme-linked immunoassays, may be exploited in themesoscale detection regions of the devices to detect a preselectedanalyte. (See Bolton et al., Handbook of Experimental Immunology, Weir,D. M., Ed., Blackwell Scientific Publications, Oxford, 1986, vol. 1,Chapter 26, for a general discussion on immunoassays.) In oneembodiment, the analyte may be an antigen and the binding moiety may bea labelled antigen binding protein, e.g. a fluorescent labelledantibody. Alternatively a sandwich immunoassay can be performed whereina tagged binding molecule, such as a fluorescent labelled antibody, isutilized to detectably bind to an analyte/binding moiety complex formedin the detection region. An example of a sandwich immunoassay isillustrated schematically in FIGS. 10A–D, wherein the surface ofmesoscale flow channel 20 in substrate 14 is coated with an antibody 103capable of binding an analyte 104. FIGS. 10B and 10C illustrate thebinding of the analyte 104 to the antibody 103 in the flow channel.Bound analyte is then detected by the subsequent addition of afluorescent labelled antibody 105 which complexes to the bound analyteas illustrated in FIG. 10D. The fluorescent labelled complex can bedetected through a translucent window over the detection region using afluorometer.

Luminescence may be readily detected in the mesoscale flow systems ofthe devices, emitted from, e.g., a fluorescein labeled binding moiety.In one embodiment, luminescence emission may be readily detected in amesoscale flow system, e.g., using a microplate reader, including aphotomultiplier tube, or a camera luminometer. In one embodiment, theanalyte may be detected by the use of a binding moiety comprising twoantibodies capable of binding to the analyte, wherein one antibody islabeled with fluorescein, which emits light, and a second antibody islabeled with rhodamine, which absorbs light. When the rhodamine andfluorescein-labeled antibodies each bind to the analyte, a quenching ofthe fluorescein can be observed, indicating the presence of the analyte.Nakamura et al., eds., Immunochemical Assays and Biosensor Technologyfor the 1990s, American Society of Microbiology, Washington, D.C., pp.205–215. In one embodiment, the fluorescein labeled antibody isimmobilized in the detection region. The analyte and therhodamine-labeled antibody are then delivered to the detection region,and quenching of the fluorescein is observed indicating the presence ofthe analyte. In another embodiment, fluorescein-labeled antibodiesconjugated to and coating a bacterial magnetic particle may be utilizedin an immunoassay, wherein the antibody is capable of binding to theanalyte. Nakamura et al. Anal. Chem. 63:268–272 (1991). In thisembodiment, the agglutination of bacterial magnetic particles conjugatedto the fluorescein-labeled antibody causes a fluorescence quenching,indicating a positive assay for the analyte. The agglutination andresulting quenching may be enhanced by applying a magnetic field to themesoscale detection region, e.g., via a magnetic source located in anappliance used in combination with the appliance.

In another embodiment, polynucleotide hybridization assays known in theart may be performed (Maniatis et al., Molecular Cloning: A LaboratoryManual, 2nd ed., Cold Spring Harbor Press, 1989). As illustratedschematically in FIG. 11, the surface of flow channel 20 in substrate 14may be coated with a polynucleotide probe 110. Upon binding of thecomplementary analyte polynucleotide 104 to the immobilizedpolynucleotide probe 110, a second detectable, e.g., fluorescentlabelled, macromolecular probe 105 can be added to bind to the samplepolynucleotide. Detection of fluorescence indicates a positive assay.

In other embodiments, the mesoscale flow system may include a chamberfor separating a selected cell population from a biological fluid samplein preparation for downstream analysis of either a macromolecule on orwithin the cells or of a component in the extracellular fluid. Themesoscale separating region includes immobilized binding moietiescapable of reversibly binding a target cell via a characteristic cellsurface molecule such as protein. The mesoscale dimension of theseparation region kinetically enhances binding of the cell and thebinding moiety. In one embodiment, the cells remain immobilized whileextracellular fluid fluid flows downstream and is analyzed. In another,flow may be continued to wash the cells, e.g., with a flow of buffer. Athigher flow rates and pressures, the washed cells are released from theseparation region and move downstream for analysis.

The devices of the invention also may include cell lysing means in fluidcommunication with the mesoscale flow channel to allow the cells to belysed prior to analysis for an intracellular molecule such as an mRNA.As illustrated in FIG. 13, the cell lysing means may comprise cellmembrane piercing protrusions 124 extending from a surface of a flowchannel 20. As fluid flow is forced through the piercing protrusion 124,cells are ruptured. Cell debris may be filtered off and intracellularanalytes may then be analyzed. Sharp edged pieces of a material such assilicon also may be utilized, trapped with the mesoscale flow system toimplement lysis of cells upon the applications of sufficient flowpressure. In another embodiment, the flow channel may simply comprise aregion of restricted cross-sectional dimension which implements celllysis upon application of sufficient flow pressure. These devicestypically are used in connection with an appliance which includes means,such as a pump, for forcing the cell containing sample into the celllysis means to cause cell lysis upon application of sufficient flowpressure. In addition, the cell lysis means may comprise a cell lysisagent. Cell lysing agents known in the art may be utilized.

As illustrated in FIG. 12, the surface of a flow channel 20 may alsoinclude protrusions 122 constituting a cellular sieve for separatingcells by size. As cell samples are flowed, typically under low pressure,through the flow channel, only cells capable of passing between theprotrusions 122 are permitted to flow through in the flow channel.

The mesoscale devices also may be utilized to implement enzymaticreactions. Mesoscale enzyme reaction chambers fabricated in thesubstrate may be temperature controlled to provide optimal temperaturesfor enzyme reactions. Inlet ports may be provided, in fluidcommunication with the enzyme reaction chamber, to allow reagents andother required enzyme assay components to be added or removed. The assaydevices embodying such chambers may be nested in an appliance such asappliance 50, illustrated schematically in FIG. 5, having means toregulate the temperature of the enzyme reaction chambers and to deliveror recover assay components through flow channels 56 in appliance 50 andports 16 in device 10. The appliance may be utilized to implement thetimed addition of sample or reagent fluids to the devices. In order toregulate the temperature of the reaction chambers, the devices may beutilized in a nesting site in an appliance utilized in combination withthe device. An electrical heating or cooling element may be provided inthe nesting site for heating or cooling the reaction chamber in thedevice. Alternatively, electrical contacts may be provided in thesubstrate and may be mated with electrical contacts in the appliance toprovide electrical resistance heating or cooling of the reactionchamber.

In one embodiment, polymerase chain reaction (PCR) may be performed in amesoscale reaction chamber to enable the detection of a polynucleotidein a sample. Inlet ports in fluid communication with the reactionchambers permit addition of required reagents, such as nucleic acids,primers and Taq polymerase. The chain reaction may be performed,according to methods established in the art (Maniatis et al., MolecularCloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press,1989). One embodiment, illustrated in FIG. 15, is PCR chip 10 whichcontains a pair of mesoscale PCR reaction chambers 164 and 166,microfabricated in a silicon substrate 14. A solution containing thepolynucleotide to be amplified is delivered from inlet 16A through flowpath 20 to the reaction chambers 164 and 166, which are heated, e.g.,electrically, to 94° C. and 65° C., respectively. A pump is attached viaport 16B to enable cycling of fluid between chamber 164, wherepolynucleotide dehybridization occurs, and chamber 166, wherepolymerization occurs. Port 16C can be used to vent the system, and alsooptionally to deliver Taq polymerase, nucleoside triphosphates, primers,and other reagents required for the polymerase reaction. A detectionchamber 22 is provided in the mesoscale flow system, containing alabelled binding moiety, such as a labeled polynucleotide probeimmobilized on a bead, to detect the presence of the amplifiedpolynucleotide product.

In operation, the PCR chip 10 is used in combination with an appliance,such as appliance 50, shown in FIG. 5, which contains a nesting site forholding the chip. The appliance 50 is provided with flow paths 56 matedto ports 16A–D. The appliance also includes valves that allow the ports16A–D to be mechanically opened and closed. The appliance 50 is used todeliver a biological sample fluid to inlet port 16A through filter 168to reaction chambers 164 and 166. Reagents such as primers, nucleosidetriphosphates, and Taq polymerase may be added to the polynucleotidesample before delivery through inlet port 16A, or optionally, reagentsmay be delivered to the sample in sample chambers 164 and 166 via port16C by means of the appliance. After delivery of a sample to PCRreaction chambers 164 and 166, the appliance is utilized to shut ports16A and 16D. Port 16C remains open as a vent. A pump disposed inappliance 50 is then utilized to cycle fluid between chamber 164, heatedto 94° C., for polynucleotide dehybridization, and chamber 166, heatedto 65° C., for the polymerization reaction.

The temperature of chambers 164 and 166 can be controlled by means of,e.g., an electrical contact integrated in the substrate below thereaction chambers, which can mate with electrical contacts in theappliance. Alternatively, an optical laser may be used to heat thereaction chambers, e.g., through a glass cover disposed over thesubstrate, or through a translucent region of the substrate itself. Whenthe polymerase cycling reaction is complete, ports 16A and 16C areclosed, port 16D is opened, and the reaction products are delivered todetection chamber 22, which contains a labeled polynucleotide probe,e.g., a probe immobilized on a fluorescent bead. Polymerization productis detected by observing the agglutination of the labeled probe and thepolymerized polynucleotide product, e.g., visually through a translucentcover disposed over the detection region. Methods and apparatus formesoscale PCR analyses are described in U.S. Ser. No. 07/877,662, thedisclosure of which is incorporated herein by reference.

In another embodiment, the devices may be utilized to perform an enzymereaction in which the mixing and addition of sample and reagentcomponents is timed, as is illustrated in the device 10 shownschematically in FIG. 21. The substrate 14 of device 10 ismicrofabricated with inlet ports 16, flow channels 20, the reactionchambers 22A and 22B and the detection chamber 22C. The reactionchambers 22A and 22B each comprise a tortuous mesoscale flow channel.The path length of the tortuous channel can be designed to permit thetimed mixing and addition of sample and reagent components. The devicemay be utilized in combination with an appliance with ports mated toports in the device, capable of delivering and receiving fluids throughthe flow system of the device, and optionally, capable of opticallydetecting a positive result in the detection chamber. In one embodiment,the cholesterol content of a sample may be assayed. Cholesterol esteraseis applied via inlet port 16A, and buffer and sample are added via inletports 16B and 16C. The mixture then flows through channel 20D to thetortuous mixing/reaction channel 22A. The time of mixing and reactionmay be predetermined by microfabricating the tortuous channel with theappropriate length. Cholesterol oxidase is added via port 16D and flowsthrough channel 20G to the tortuous channel 22B, where the timed mixingand reaction of the oxidase with the fluid from channel 22A occurs. Apositive result can be detected optically by observing the detectionchamber 22C through an optical window disposed over the substrate. Thedetection chamber 22C may be provided with a binding moiety capable ofdetectably reacting with the product of the enzyme reaction. The devicemay be applied to a range of clinical enzymatic and other reactions.

Optionally, depending on the protocol being exploited in the structureof the chip, the appliance may also be designed to inject reagentsnecessary to complete the assay, e.g., inject a binding protein taggedwith an optically detectable moiety, a substrate solution, or otherreagents. The pressure of fluid flow in the mesoscale flow channel 20 indevice 10 can be detected by the pressure detectors 54 provided inappliance 50. A microprocessor may be included in the appliance toassist in the collection of data for one or a series of assays. In orderto enhance the accuracy of an assay, the substrate may be fabricated toinclude a control region in the flow system, e.g., a region which doesnot include binding moieties, such that the sample is directed to boththe detection and control regions. Data obtained from sample fluidflowing through the control region may be detected and compared with thedata from the sample detection region to increase the precision of theassay.

It will be understood that the above descriptions are made by way ofillustration, and that the invention may take other forms within thespirit of the structures and methods described herein. Variations andmodifications will occur to those skilled in the art, and all suchvariations and modifications are considered to be part of the invention,as defined in the claims.

The invention will be understood further from the following nonlimitingexamples.

EXAMPLE 1

Capillary agglutination of red blood cells and immobilized anti-Aantiserum was examined in a series of silicon substrates 14 (shownschematically in FIG. 6), fabricated with flow channels 20 of varyingwidth. The silicon substrates 1–5 included flow channels 20A and B witha depth of 10 μm and widths ranging from 20 to 300 μm (Table 2). Theinside surface of the channels were coated with anti-A (1:10 dilution)by first filling the channel with the antibody (capillary action) andallowing it to dry. A Type blood (diluted 1:5) was then introduced intochannel 20 from inlet port 16 by capillary action and the channel wasobserved visually using a microscope (Leitz Aristomet). Results aresummarized in Table 2.

TABLE 2 DEPTH CHANNEL WIDTH SUBSTRATE # (μm) (μm) AGGLUTINATION 1 10A:20  + B:40  + 2 10 A:60  + B:80  + 3 10 A:100 + B:120 + 4 10 A:150 +B:200 + 5 10 A:250 + B:300 +

EXAMPLE 2

A plastic-silicon hybrid was fabricated by attaching a plastic (3Mtransparency sheet) cover over the silicon substrate 14, which ismicrofabricated with flow channels 20 with entry ports 16 on oppositesides of the channel and a central detection chamber 22 (shownschematically in FIG. 7). A dilution of anti-A (in 0.05 M sodiumbicarbonate pH 9.6) and a 1:10 dilution of Type A blood in saline wereintroduced via syringe using a holder into the entry ports 16 onopposite ends of the channel 20. The solutions mixed together in thecentral chamber 22 and agglutination was observed through the plasticcover by light microscopy. The results are summarized in Table 3.

TABLE 3 AGGLUTINATION IN ANTI-A DILUTION CHANNEL Gamma Kit 1:20  + GammaMurine Mono 1:20  + Gamma Human Dilution 1:5  + Immucor Affinity pure1:100 + Immucor Ascites 1:100 +

EXAMPLE 3

A plastic-silicon hybrid was fabricated by attaching a piece of plastic(3M transparency sheet) over a silicon substrate 14 etched with amesoscale flow channel 20 having entry ports 16 microfabricated onopposite sides of the channel and a central mesoscale mixing chamber 22(shown schematically in FIG. 7). A solution of mouse IgG (50 μg/mL in0.05 M sodium bicarbonate pH 9.6) (SIGMA Cat. no. 1–5381) and a 1:20dilution of goat anti-mouse IgG (H&L)—fluorescence carboxylate beads(Polysciences, Inc.) in PBS buffer were introduced via syringe using aholder into the entry ports on opposite ends of the channel. Thesolutions mixed together in the central chamber 22 and agglutination wasobserved through the transparent plastic cover by light microscopy.

EXAMPLE 4

An analytical element 14 having three pairs of mesoscale analyticalchambers 22A–C, linked to three pairs of mesoscale control chambers24A–C emanating from entry port 16 is used for the determination of theblood group of a blood sample (shown schematically in FIG. 8). Thesurface of chamber 22A is sensitized with antibody to blood group Aantigen, chamber 22B is sensitized with antibody to blood group Bantigen and chamber 22C is sensitized with antibody to Rhesus antigen.The surface of chambers 24A, 24B and 24C are untreated and used asnegative controls. A finger prick sample of blood is drawn into thedevice through port 16 using a syringe. Binding of red cells to thesurface of the three chambers 22A–C is observed. The presence of redcells on the surface of a particular chamber (22A, 22B and/or 22C)denotes a positive result for the blood group antigen. The analyticaldevice containing the sample is then discarded.

EXAMPLE 5

An analytical element 14 (shown schematically in FIG. 9) having threechambers, 22A, 22B and 22C, linked by channels 20 emanating from inletport 16 is used for the determination of the blood group of a bloodsample. Chamber 22A contains beads sensitized with antibody to bloodgroup A antigen, chamber 22B contains beads sensitized with antibody toblood group B antigen, and chamber 22C contains beads sensitized withantibody to Rhesus antigen. A finger prick sample of blood is drawn intothe device using a syringe. Binding of red cells to the beads andsubsequent agglutination in the chambers is observed. The presence ofagglutinated red cells in a particular chamber denotes a positive resultfor the blood group antigen. The analytical device containing the sampleis then discarded.

EXAMPLE 6

An analytical element 14 (shown schematically in FIG. 8) having threepairs of chambers 22A, 22B and 22C linked by channels 20 emanating fromentry ports 16 is used for the determination of the blood group of ablood sample. The element also includes the control chambers 24A, 24Band 24C. The surface of chamber 22A is sensitized with antibody to bloodgroup A antigen, chamber 22B is sensitized with antibody to blood groupB antigen, and chamber 22C is sensitized with antibody to Rhesusantigen. The surface of chambers 24A–C are untreated and act as negativecontrols. A finger prick sample of blood is mixed with a fluorescent dyeand then drawn into the inlet port 16 using a syringe. Binding of thefluorescent red cells to the surface in the three chambers (22A, 22B,and/or 22C) is rapidly observed using a microfluorometer and denotes apositive result for the blood group antigen. The analytical devicecontaining the sample is then discarded.

EXAMPLE 7

The growth of an organism is monitored in the device shown schematicallyin FIG. 4. The fractal pattern of mesoscale flow paths 40 in thesubstrate 14 are filled via inlet port 16A with 2 μL of a mixture ofgrowth medium which has been inoculated with a sample of a testspecimen. The device is sealed and incubated for 60 minutes at 37° C.Growth is detected by visual inspection using a microscope or bydetermining the flow properties of the channel system, e.g., via theelectrical conductivity probe 17. The absence of flow indicates growthand consequent blockage of the channel system.

EXAMPLE 8

Sperm functions are tested on the microfabricated solid substrate 14shown in FIG. 14. A sperm sample is added to the inlet port 16 and thenflows through the mesoscale flow channel 20 to the detection chambers22A–D each having a reagent addition port 140. Detection chamber 22Aprovides a test for leucocytes and includes beads containing immobilizedantibody to common leukocyte antigen. Detection chamber 22B provides atest for sperm antibodies and contains beads on which are immobilizedantibody to human IgG (e.g., Bio-Rad, Immunobead Cat. No. 170-5100).Chamber 22C provides a test for acrosome reaction and containsfluorescein labeled lectin. Chamber 22D provides a test forsperm-cervical interaction and contains hyaluronic acid or bovinecervical mucus. Agglutination in the chambers may be detected eitheroptically manually or by machine. The fractal pattern of flow channels40 is used to test flow properties of the sample. The distance that thesperm sample travels along the fractal flow path serves as an indicatorof sperm motility. Alternatively, mesoscale flow systems fabricated withother configurations may be utilized, such as a nonbranching flowchannel.

EXAMPLE 9

A polymerase chain reaction is performed in the device illustratedschematically in FIG. 15, to detect the presence of a polynucleotide ina fluid sample. The device 10 shown in FIG. 15 includes a solidsubstrate 14 microfabricated with inlet ports 16A–D connected to themesoscale flow channel 20. Mesoscale flow channel 20 also is providedwith PCR reaction chambers 164 and 166, filters 168 and detectionchamber 22. The device 10 is used in combination with an appliance, suchas appliance 50 in FIG. 5, that is provided with fluid channels, a fluidpump and temperature control elements for controlling the temperature ofreaction chambers 164 and 166. The appliance also includes fluid flowpaths with valves in fluid communication with ports 16A, 16B, 16C, and16D allowing the ports to be reversibly opened or closed during anassay.

To perform a PCR analysis to detect a polynucleotide in a cell, a samplecell lysate is added to a buffered solution of Taq polymerase,nucleoside triphosphates, polynucleotide primers and other reagentsrequired for a PCR assay. The cell sample lysate is delivered via theappliance through entry port 16A to PCR reaction chambers 164 and 166.Ports 16A and 16D are closed by means of valves included in theappliance, while port 16B and 16C are open. Means such as electricalmeans are included in the appliance to regulate the temperature of thereaction chambers 164 and 166. A pump in the appliance connected throughport 16B is used to cycle sample fluids between reaction chamber 164,set at 94° C., for polynucleotide dehybridization, and reaction chamber166, set at 65° C., for polymerase reaction. Port 16C serves as a vent.After the polymerase chain reaction is complete, port 16C is closed and16D is opened and the pump in the appliance connected to port 16B isused to deliver the sample from the PCR reaction chambers 164 and 166 tothe detection chamber 22. Detection chamber 22 is provided withfluorescent labeled beads on which are immobilized polynucleotide probescapable of binding the amplified polynucleotide. The agglutination ofthe amplified polynucleotide with the labeled polynucleotide probe isdetectable through a window disposed over the detection region 22 andprovides a test for the presence of amplified polynucleotide product.

EXAMPLE 10

A multitest device 10 including substrate 14, shown schematically inFIG. 16, is used to detect the presence of an intracellularpolynucleotide in a biological cell-containing fluid sample. The deviceis used in combination with an appliance, such as appliance 50, shown inFIG. 5. The appliance includes fluid channels with ports, that includevalves that may be reversibly opened and closed, mated to the ports indevice 10, allowing the ports in the device to be mechanically openedand closed during an assay. The appliance also includes means, such aselectrical contacts mated to contacts imbedded in the substrate 14, forregulating the temperature of reaction chambers 164 and 166. Theappliance further includes a pump to control the flow of fluid throughthe device 10.

Initially, the valves in the appliance are used to close ports 16C and16D, while ports 16A and 16B remain open. The sample is directed to thesample inlet port 16A by a pump in the appliance, and flows through themesoscale flow path 20A to chamber 22A, which contains binding moietiesimmobilized on the wall of the chambers for selectively binding to asurface molecule on a desired cell population. After binding of thedesired cell population in chamber 22A, flow with buffer is continued,exiting through port 16B, to purify and isolate the cell population.Port 16B is then closed and port 16C is opened. Flow is then increasedsufficiently to dislodge the isolated cells from the surface of chamber22A to chamber 22B where membrane piercing protrusions 124 in chamber22B tear open the cells releasing intracellular material.

Sample flow continues past filter 168, which filters off large cellularmembranes and other debris, to the mesoscale PCR chambers 164 and 166.The valves in the appliance are used to open port 16B and to close port16A. Taq polymerase, primers and other reagents required for the PCRassay are added to chambers 164 and 166 through port 16C from a matedport and flow path in the appliance. A pump in the appliance connectedvia port 16B is used to cycle the PCR sample and reagents betweenchambers 164 and 168, set at 94° C. and 65° C. respectively, toimplement a polynucleotide dehybridization and polymerization cycle,allowing the production and isolation of product polynucleotide. Thevalves in the appliance are used to close port 16C and to open port 16D.The pump in the appliance connected to port 16B is used to direct thepolymerized polynucleotide isolated from the cell population to thefractal detection region 40, which contains immobilizing bindingmoieties, such as a complementary polynucleotide probe. Flow restrictionin the fractal region 40 indicates a positive assay for theintracellular polynucleotide.

EXAMPLE 11

A chemiluminescent peroxyoxylate organic phase reaction was conducted ina mesoscale flow channel. A Cyalume™ light stick (Aldrich, Milwaukee,Wis.) was opened and the mixture of peroxyoxylate and fluorophore(component A) were drained into a test tube. The glass vial containingthe oxidant was removed and washed with alcohol. The contents of thevial (component B) were transferred to a test tube. A 100 μL sample ofcomponent A and 50 μL of component B were mixed together to initiate thechemiluminescent reaction.

A sample of the fluorescent solution was introduced into the centralinlet port of chip #6, provided with a chamber with dimensions of 812 μmin width, 400 μm in depth and 5.2 mm in length, connected to two 20 μmdeep, 100 μm wide, 3.25 mm long channels. Any excess sample was wipedoff the surface of the chip, and the chip was placed into a modifiedmicrowell strip holder. The light emission from the mesoscale flowchannel was measured using an Amerlite microplate reader (AmershamDiagnostics Ltd., Amersham, UK). A similar experiment was performedusing a 300 μm wide, 20 μm deep mesoscale flow channel (volume 70.2 μL)in chip #5. Light emission (peroxyoxylate chemiluminescence) wasdetected and measured in units of RLU (relative light units) from themesoscale flow channels in the different chips using the luminescencemicroplate reader (Table 4).

TABLE 4 Channel Light Chip Volume Emission (RLU) #6  1702 pL 718.26 #5 70.2 pL 35.63

EXAMPLE 12

In an aqueous phase reaction, the chemiluminescent horseradishperoxidase catalyzed oxidation of isoluminol was examined. Theluminol-hydrogen peroxide reagent was prepared as follows: Sodiumluminol (12.5 mg) was dissolved in 50 mL of Tris buffer (0.1 mol/L, pH8.6). 15.5 μL of hydrogen peroxide (30% w/v) was mixed with 0.5 mL ofTris buffer (0.1 mol/L, pH 8.6). These two solutions were combined andprotected from light. The luminol-hydrogen peroxide reagent (100 μL), 5μL of 4-iodophenol ((Aldrich) (1 mg/ml in 0.1 mol/L Tris buffer, pH8.6), and 10 μL of a dilution of horseradish peroxidase (Type VIA, 1mg/mL) in Tris buffer (0.1 mol/L, pH 8.6) were mixed together. A sampleof this solution was introduced into the central chamber of chip #6 orinto the 300 μm channel of chip #5. The light emission was then measuredusing the Amerlite microplate reader.

The chemiluminescence emission from the horseradish peroxidase catalyzedoxidation of luminol in the different mesoscale channels was detectedusing the luminescence microplate reader. A peroxidase assay usingdilutions of the stock peroxidase gave a dose dependent relationship(Table 5).

TABLE 5 Light Channel Peroxidase Emission Chip Volume dilution (RLU) #6 1702 pL undiluted  0.18* 1:10  4.68 1:100  2.23 1:1000 1.82 #5  70.2 pLundiluted 2.09 *Low light level because of substrate exhaustion.

EXAMPLE 13

Chemiluminescent reactions in the mesoscale flow channels were detectedphotographically. Mesoscale channels of chip #6 were filled with theperoxyoxylate or horseradish peroxidase (10 μg/mL)-luminol-peroxidereaction mixtures as described in Examples 11 and 12. The light emissionwas detected by contacting the chip with instant photographic film(Polaroid, Type 612) in a camera luminometer (Wolfson AppliedTechnology, Birmingham, UK). Light emission from the differentchemiluminescent reactions in the mesoscale flow channels was detectedusing high speed instant photographic film (Table 6). The lower lightintensity from the peroxidase reaction required a longer exposure time.

TABLE 6 Detected (D) Exposure Time Not Detected (ND) Peroxyoxylate  1second D reaction  5 minutes* D Horseradish 10 minutes D peroxidasereaction *After 2 day incubation at room temperature.

EXAMPLE 14

An experiment testing different spermicides using a mesoscale flowsystem was conducted. A chip comprising two chambers (5.2 mm long, 750μm wide, 1.5 mm deep) each linked at each end to an entry hole by achannel (3.25 mm long, 100 μm wide, 20 μm deep) was used for thesimultaneous testing of the spermicidal activity of nonoxynol-9 andC13-G (Biosyn, Inc., PA). The four channels were filled with HTF-BSAsolution (channel #1, control), 0.005% (channel #2), 0.0125% (channel#3), and 0.05% (channel #4) nonoxynol-9 (or C13-G), respectively. Asample of semen was placed in each chamber, and the progress of sperminto the adjoining channels monitored using the microscope. The numberof sperm observed in the channels was in the following order ofdecreasing sperm count: channel #1>#2>#3>#4. The most sperm were seen inthe control channel, and none were seen in channel #4 which containednonoxynol-9 or C13-G at the optimum concentration for spermicidalaction.

EXAMPLE 15

The interaction of a sperm sample with cervical mucus in a mesoscaleflow system was tested in a chip comprising two chambers (5.2 mm long,750 μm wide, 1.5 mm deep) each linked at each end to an entry hole by achannel (3.25 mm long, 100 μm wide, 20 μm deep). The channels werefilled with HTF-BSA solution and a cervical mucus sample (collected atapproximately day 14 of the patient's menstrual cycle) placed in each ofthe central chambers. Sperm did not migrate into the cervical mucus andthose that penetrated died, as anticipated because cervical mucus isknown to be hostile to sperm at this time during the menstrual cycle.Moghissi et al., Am. J. Obstet. Gynecol., 114:405 (1972).

EXAMPLE 16

A test of the interaction of hyaluronic acid with a sperm sample wasconducted to assess the cervical interaction properties of a spermsample. The test was conducted in a chip comprising two chambers (5.2 mmlong, 750 μm wide, 1.5 mm deep) each linked at each end to an entry holeby mesoscale flow Channels #1, #2, #3 and #4 (3.25 mm long, 100 μm wide,20 μm deep). Channel #1 was a control channel. Channels were filled withHTF-BSA solution and solutions of hyaluronic acid (Sigma) in HTF-BSA(channels #2, #3, #4, 5 mg/mL, 2.5 mg/mL, and 1.3 mg/mL, respectively).A semen sample was placed in each of the central chambers. Sperm did notmigrate into channel #2, containing 5 mg/mL hyaluronic acid, but theextent of migration increased as the concentration of hyaluronic aciddecreased in channels #3 and #4.

EXAMPLE 17

An immunobead test for the presence of IgG antibodies in a sperm samplewas conducted. Immunobeads (BioRAD, Richmond, Calif.), microbeads coatedwith an antibody to human IgG, were diluted to 1 mg/mL in HTF-BSAsolution (Irvine Scientific, Santa Ana, Calif.). A microchannel (250 μmwide, 20 μm deep, and 10 mm long) in a glass-silicon chip was filledwith a sample of the immunobead solution and a semen sample (ca 1.2 μL)was applied to the channel entry. Agglutination of sperm by theimmunobeads due to the presence of antibodies in the sperm sample wasobserved in the channel. As a control, the experiment was performed on aglass microscope slide using larger volumes of the immunobead reagentand semen sample, and this was also positive (agglutination observed).

It will be understood that the above descriptions are made by way ofillustration, and that the invention may take other forms within thespirit of the structures and methods described herein. Variations andmodifications will occur to those skilled in the art, and all suchvariations and modifications are considered to be part of the invention,as defined in the claims.

1. A device comprising a first polymer substrate layer having aplurality of mesoscale channels fabricated thereon, which first polymersubstrate layer is overlaid by a cover layer, which cover layercomprises a transparent portion, whereby the plurality of channels aresealed between the first polymer substrate layer and the cover layer. 2.The device of claim 1, wherein the cover layer and the first substratelayer are adhered.
 3. The device of claim 1, wherein the first polymersubstrate is formed by molding the polymeric substrate.
 4. The device ofclaim 1, wherein the cover layer comprises glass.
 5. The device of claim1, wherein the cover layer comprises a material other than glass.
 6. Thedevice of claim 1, wherein the cover layer comprises a plastic.
 7. Thedevice of claim 1, wherein the cover layer comprises a plastic sheet. 8.The device of claim 1, wherein the flow channels are fabricated on thefirst substrate by at least one of spin coating and vapor deposition,photolitography, wet chemical etching and plasma processing.
 9. Thedevice of claim 1, further comprising a second cover layer bonded to thefirst substrate.
 10. The device of claim 1, wherein the plurality ofchannels intersect at a plurality of intersections.
 11. The device ofclaim 1, wherein the channels are between about 0.5 and 500 microns inat least one channel dimension.
 12. The device of claim 1, wherein thechannels are between about 2 and 500 microns wide and between 0.1 and500 microns deep.
 13. The device of claim 1, further comprising a sampleinlet port coupled to at least one of the plurality of channels.
 14. Thedevice of claim 1, further comprising at least one source of at leastone biological material.
 15. The device of claim 14, wherein thebiological material comprises at least one of blood, plasma, serum,urine, sputum, saliva, cells and antibodies.
 16. The device of claim 14,wherein the at least one source of at least one biological fluid isconnected to a sample inlet port, which sample inlet port is coupled toat least one of the plurality of channels.
 17. The device of claim 1,further comprising an appliance, the appliance comprising a nesting sitefor receiving the first substrate when overlayed by the transparentcover layer.
 18. The device of claim 17, the appliance comprising aconduit for delivering source of material to at least one of theplurality of channels.
 19. The device of claim 17, the appliancecomprising an injector for injecting a sample into contact with at leastone of the plurality of channels.
 20. The device of claim 19, whereinthe injector is a pressure injector and the appliance further comprisesa pressure detector.
 21. The device of claim 17, wherein the cover layercomprises a sample inlet port fluidly coupled to at least one of theplurality of channels, the appliance comprising a flow line coupled tothe sample inlet port.
 22. The device of claim 17, further comprisingone or more valves in the appliance or in contact with one or more ofthe plurality of channels, which one or more valves regulates fluidflow.
 23. The device of claim 1, further comprising means for samplemovement.
 24. The device of claim 1, further comprising means forreagent movement.
 25. The device of claim 1, further comprising adetection region within at least one of the channels.
 26. The device ofclaim 1, further comprising a conductivity sensor in at least one of thechannels.
 27. The device of claim 1, further comprising a pressuresensor in at least one of the channels.
 28. The device of claim 1,further comprising an optical detector proximal to channel for detectingan optically detectable moiety within the channel.
 29. The device ofclaim 28, wherein the optical detector comprises a spectroscope.
 30. Thedevice of claim 28, wherein the optical detector comprises a microscope.31. The device of claim 28, wherein the optical detector comprises alight source.
 32. The device of claim 28, wherein the optical detectorcomprises a camera.
 33. The device of claim 28, wherein the opticaldetector detects a fluorescent or luminescent signal in a detectionregion coupled to or within a channel of the device.
 34. The device ofclaim 1, further comprising a tilting mechanism for tilting the firstsubstrate when overlaid by the cover layer.
 35. The device of claim 1,further comprising a detection region within at least one mesoscalechannel or chamber of the device, the detection region comprising apolymer bead disposed therein.
 36. The device of claim 1, furthercomprising a plurality of electrical contacts configured to resistivelyheat a portion of the first substrate.
 37. The device of claim 1,further comprising temperature control means for controlling thetemperature of a portion of the device.
 38. The device of claim 1,further comprising an inlet port and a source of a plurality of samplematerials, which, during operation of the device, are flowed through theinlet port and into at least one of the plurality of channels.
 39. Thedevice of claim 1, the device further comprising an inlet port and aplurality of source of separate sample materials, which, duringoperation of the device, are flowed through the inlet port and into atleast one of the plurality of channels.
 40. The device of claim 1,wherein the first substrate and cover layer are each between 0.2 and 2centimeters square.
 41. The device of claim 1, wherein the firstsubstrate is manufactured from a polytetrafluoroethylene.
 42. The deviceof claim 1, wherein the total volume of the plurality of channels isless than 10 microliters.
 43. The device of claim 1, wherein thechannels comprise at least one surface coating.
 44. The device of claim1, further comprising a magnetic bead disposed within one or more of theplurality of channels.
 45. The device of claim 44, further comprising amagnetic field source of directing movement of the magnetic bead. 46.The device of claim 1, wherein at least one of the channels varies inwidth along the length of the at least one channel.
 47. The device ofclaim 1, further comprising a pump coupled to a port coupled to at leastone of the plurality of channels.
 48. The device of claim 1, furthercomprising reagents for PCR amplification within the channels of thedevice.
 49. The device of claim 1, further comprising a microprocessorcoupled to a detector which is mounted proximal to the cover layer. 50.The device of claim 1, wherein the plurality of channels comprise atleast one sample and at least one control region.
 51. The device ofclaim 1, further comprising a microprocessor coupled to a detector whichis mounted proximal at least one sample and at least one control region,which microprocessor compares data from the sample and the controlregion.